Project Summary The timely delivery of membrane-bound vesicles and tubules bearing transmembrane protein and lipid cargo to discrete cellular locations via specific trafficking pathways is fundamental to cell biology and human health. Many proteins associated with trafficking pathways are linked to serious and crippling human diseases, including neurological disorders like Alzheimer's and the hereditary spastic paraplegias. Although certain trafficking proteins and pathways are well characterized, we lack direct evidence or have only partial evidence for other pathways that we infer must exist between membranes. This constitutes an enormous gap in our current understanding of fundamental cell biology. Our goal is to elucidate the molecular structures and functions of important coat protein complexes that initiate trafficking pathways by forming coats around vesicles or tubules at specific membranes. Coat proteins recognize and package relevant cargoes, and they promote efficient assembly of additional required protein components, like SNAREs. While clathrin coats have been extensively studied, functions of certain non-clathrin coats remain virtually unknown. Increasing evidence indicates non-clathrin coats assemble using distinct mechanisms, suggesting clathrin cannot be considered a paradigm for coat assembly. We investigate non- clathrin coat complexes, including adaptor protein 4 (AP4), coat protein complex I (COPI), and retromer, by using a variety of tools to ascertain molecular mechanisms of coat assembly and regulation. Biochemical and proteomic approaches allow us to identify new components of coated structures, especially cargo molecules, accessory, and regulatory proteins. Structural methods like X-ray crystallography, NMR, and electron microscopy reveal at the molecular level how coats interact with key protein partners and allow us to map specific binding interfaces. Biophysical techniques enable us to quantify binding affinities and to probe interfaces identified in structural models. With collaborators, we use molecular data to design experiments in cultured cell lines and in model organisms to explore how a variety of protein-protein interactions drive phenotypes at the cellular and organismal levels. Ultimately, we hope to gain a molecular understanding of how non-clathrin coats assemble at distinct membranes to drive different trafficking pathways. We anticipate this work will reveal new mechanisms of cargo recognition, coat assembly, and regulation. We further aim to uncover the molecular basis of specific diseases associated with these coat proteins.